NK zygote molecules and methods of use thereof

Abstract

The name of the invention is NK zygote molecules and methods of use thereof. Compositions for activating NK cells to stimulate an immune response for the treatment of cancer and other disorders are provided. In one embodiment, the present invention provides a compound comprising an NK binding domain that binds to CD16; an NK activation domain operably linked to the NK binding domain; and a targeting domain selectively binding to a target cell and operably linked to the NK activating domain and the NK binding domain, wherein the targeting domain binds to CLEC12A.

Description

[0001] This application is a divisional application. The application date of the original application is October 17, 2019, the application number is 2019800675953, and the name of the invention is “NK engager molecules and methods of use thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Serial No. 62 / 747,983, filed on October 19, 2018, the entire contents of which are incorporated herein by reference in their entirety.

[0004] Government funding

[0005] This invention was made with government support under Grants CA111412 and CA65493 from the National Institutes of Health, Grants CA36725, CA72669, CA077598, and CA197292 from the National Cancer Institute, and Grant CA150085 from the Department of Defense. The government has certain rights in this invention.

[0006] Sequence Listing

[0007] The materials in the attached sequence listing are hereby incorporated by reference into this application. The attached sequence listing text file named GTBIO2090_1WO_Sequence_Listing.txt was created on October 15, 2019 and is 23kb. The file can be accessed using Microsoft Word on a computer using Windows OS. Background of the Invention Technical Field

[0008] The present invention relates generally to immunotherapy and, more particularly, to compositions useful for engaging natural killer (NK) cells in an immune response. Background Art

[0009] Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Like cytotoxic T cells, NK cells deliver large quantities of membrane-penetrating and apoptosis-inducing granzymes and perforin granules. Unlike T cells, NK cells do not require antigen priming and recognize targets by engaging activating receptors in the absence of MHC recognition. NK cells express CD16, an activating receptor that binds to the Fc portion of IgG antibodies and participates in antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells are regulated by IL-15, which can induce increased antigen-dependent cytotoxicity, lymphokine-activated killing activity, and / or mediate interferon (IFN), tumor necrosis factor (TNF), and / or granulocyte-macrophage colony-stimulating factor (GM-CSF) responses. All of these IL-15-activated functions contribute to improved cancer defense.

[0010] Therapeutically, for example, adoptive transfer of NK cells can induce remissions in patients with refractory acute myeloid leukemia (AML) when combined with lymphodepleting chemotherapy and IL-2 to stimulate NK cell survival and expansion in vivo. This therapy may be limited by a lack of antigen specificity and IL-2-mediated induction of regulatory T (Treg) cells, which suppress NK cell proliferation and function. Generating an agent that drives NK cell antigen specificity, expansion, and / or persistence while bypassing the negative effects of Treg suppression could enhance NK cell-based immunotherapy. Summary of the Invention

[0011] The present invention relates to compounds and compositions for activating NK cells to stimulate an immune response for the treatment of cancer and other disorders. In one embodiment, the present invention provides a compound comprising an NK engaging domain; an NK activation domain operably linked to the NK engaging domain; and a targeting domain that selectively binds to a target cell and is operably linked to the NK activation domain and the NK engaging domain, wherein the targeting domain selectively binds to CLEC12A.

[0012] In some embodiments, the NK engaging domain comprises a portion that selectively binds to CD16. In some embodiments, the NK engaging domain portion comprises an antibody or binding fragment thereof or a nanobody, also known as a single domain antibody (sdAb or VHH). In some embodiments, the antibody binding fragment comprises scFv, F(ab)2, or Fab. In some embodiments, the antibody or binding fragment thereof or nanobody is human or humanized. In some embodiments, the antibody or binding fragment thereof or nanobody is of the camelid family.

[0013] In some embodiments, the NK activation domain comprises a cytokine or a functional fragment thereof. In some embodiments, the NK activation domain comprises IL-15 or a functional fragment thereof. In some embodiments, the IL-15 comprises the amino acid sequence of SEQ ID NO: 9 or a functional variant thereof. In one aspect, the functional variant of IL-15 comprises an N72D or N72A amino acid substitution compared to SEQ ID NO: 9.

[0014] In some embodiments, the targeting domain portion comprises an antibody or its binding fragment or nanobody. In some embodiments, the antibody binding fragment comprises scFv, F(ab)2 or Fab.

[0015] In some embodiments, the NK engaging domain comprises a moiety that selectively binds to CD16, the NK activation domain comprises IL-15, and the targeting domain selectively binds to CLEC12A.

[0016] In some embodiments, the compounds and compositions described herein comprise at least one flanking sequence connecting the two domains. In some embodiments, the compounds and compositions described herein further comprise a second flanking sequence connecting the two connected domains to the third domain. In some embodiments, the flanking sequences flank the NK activation domain. In some embodiments, the first flanking sequence is the C-terminus of the NK engagement domain, and the second flanking sequence is the N-terminus of the anti-CLEC12A targeting domain.

[0017] In some embodiments, provided herein is an isolated amino acid sequence comprising SEQ ID NO.: 1. In some embodiments, provided herein is an isolated DNA sequence encoding the amino acid sequence of SEQ ID NO.: 1.

[0018] In some embodiments, provided herein is an isolated amino acid sequence comprising SEQ ID NO.: 2. In some embodiments, provided herein is an isolated DNA sequence encoding the amino acid sequence of SEQ ID NO.: 2.

[0019] In some embodiments, provided herein is an isolated amino acid sequence comprising SEQ ID NO.: 4. In some embodiments, provided herein is an isolated DNA sequence encoding the amino acid sequence of SEQ ID NO.: 4.

[0020] In some embodiments, provided herein are compositions comprising a compound described herein and a pharmaceutically acceptable carrier.

[0021] In some embodiments, provided herein are methods comprising: administering to a subject a compound described herein in an amount effective to induce NK-mediated killing of a target cell. In some embodiments, the target cell is a cancer cell.

[0022] In some embodiments, provided herein are methods for stimulating the expansion of NK cells in vivo, the methods comprising: administering to a subject a compound described herein in an amount effective to stimulate the expansion of NK cells in the subject.

[0023] In some embodiments, provided herein are methods for treating cancer in a subject, comprising administering to the subject an amount of a compound described herein effective for treating cancer. In some embodiments, the cancer comprises prostate cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, melanoma, kidney cancer, renal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, or hematopoietic cancer. In some embodiments, provided herein are methods further comprising administering the compound before, simultaneously with, or after chemotherapy, surgical resection of the tumor, or radiotherapy. In some embodiments, chemotherapy comprises hexamethonium, amsacrine, L-asparaginase, L-asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytosine, cytarabine, dacarbazine, actinomycin D, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, flu In some embodiments, the hematopoietic cancer is AML. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figures 1A-1C Shown is the proliferation of NK cells after treatment with CLEC12A TriKE ( Figure 1A ), NK cell killing ( Figure 1B ) and functional assays ( Figure 1C ).

[0025] Figure 2 Shown are the percentages of CD33 and CLEC12A surface expression on primary AML samples from 10 patients.

[0026] Figures 3A-3B CD16-IL15-CLEC12A TriKE ( Figure 3A ) and mechanism of action ( Figure 3B).

[0027] Figure 4 Binding of the CD16-IL15-CLEC12A TriKE to a CLEC12A-expressing target is shown.

[0028] Figures 5A-5B Shown is the CD16-IL15-CLEC12A TriKE promotion of NK cell proliferation.

[0029] Figures 6A-6C CD16-IL15-CLEC12A TriKE-induced degranulation of AML target cells is shown ( Figure 6A ) and cytokine production ( Figure 6B -C).

[0030] Figures 7A-7B CD16-IL15-CLEC12A TriKE induction of AML target cell killing is shown.

[0031] Figures 8A-8D Shown is CD16-IL15-CLEC12A TriKE-induced killing of primary AML targets in vitro.

[0032] Figures 9A-9C Shown is CD16-IL15-CLEC12A TriKE induction of NK cell proliferation.

[0033] Figures 10A-10D Functional validation of the CD16-IL15-CLEC12A TriKE is shown.

[0034] Figures 11A-11C Shown is CD16-IL15-CLEC12A TriKE induction of target cell killing in a live imaging assay. THP-1 tumor target is shown.

[0035] Figures 12A-12G Shown is CD16-IL15-CLEC12A TriKE-induced killing of primary AML blasts.

[0036] Figures 13A-13G It was shown that the CD16-IL15-CLEC12A TriKE restricted tumor growth in vivo.

[0037] Figures 14A-14C Binding validation of CD16-IL15-CLEC12ATriKE is shown.

[0038] Figures 15A-15B Shown is CD16-IL15-CLEC12A TriKE induction of target cell killing in a live imaging assay. HL-60 tumor target is shown.

[0039] Figures 16A-16B CD16-IL15-CLEC12ATriKE-mediated target killing is shown. Target gating strategy is shown ( Figure 16A ) and target cell killing ( Figure 16B ). AML blast target is shown.

[0040] Figure 17 Shown is a gating strategy to identify cancer stem cells in bone marrow samples from AML patients.

[0041] Figures 18A-18B Shown is the expression of CLEC12A and CD33 within the CD34pos progenitor cell compartment in the bone marrow. Cell populations from two representative donors are shown ( Figure 18A ) and cell colonies after treatment with the indicated TriKE ( Figure 18B ).

[0042] Figure 19 Shown is the gating strategy for determining different CD34pos progenitor cell subsets in healthy bone marrow samples. DETAILED DESCRIPTION

[0043] Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Like cytotoxic T cells, NK cells deliver large quantities of membrane-penetrating and apoptosis-inducing granzymes and perforin granules. Unlike T cells, NK cells do not require antigen priming and recognize targets by engaging activating receptors in the absence of MHC recognition. NK cells express CD16, an activating receptor that binds to the Fc portion of IgG antibodies and participates in antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells are regulated by IL-15, which can induce increased antigen-dependent cytotoxicity, lymphokine-activated killing activity, and / or mediate interferon (IFN), tumor necrosis factor (TNF), and / or granulocyte-macrophage colony-stimulating factor (GM-CSF) responses. All of these IL-15-activated functions contribute to improved cancer defense.

[0044] Therapeutically, for example, adoptive transfer of NK cells can induce remissions in patients with refractory acute myeloid leukemia (AML) when combined with lymphodepleting chemotherapy and IL-2 to stimulate NK cell survival and expansion in vivo. This therapy may be limited by a lack of antigen specificity and IL-2-mediated induction of regulatory T (Treg) cells, which suppress NK cell proliferation and function. Generating an agent that drives NK cell antigen specificity, expansion, and / or persistence while bypassing the negative effects of Treg suppression could enhance NK cell-based immunotherapy.

[0045] The present disclosure describes the generation of trispecific molecules comprising two domains capable of driving NK cell-mediated killing of tumor cells (e.g., CD33+ and / or CD33- tumor cells) and an intramolecular NK activation domain capable of generating NK cell self-sustaining signals. The trispecific molecules can drive NK cell proliferation and / or enhance NK cell-driven cytotoxicity against, for example, HL-60 targets, cancer cells, or cancer cell-derived cell lines.

[0046] The present invention is based on the development of a CD16 / IL-15 / CD33 trispecific killer engager (TriKE) molecule that uses natural killer (NK) cells to target acute myeloid leukemia (AML) cells. The molecule contains an anti-CD16 camelid nanobody that activates NK cells, an anti-CD33 single-chain variable fragment (scFv) that binds to the cancer target, and an IL-15 molecule that drives NK cell priming, expansion, and survival. Using an early version of this molecule, the CD33 TriKE was shown to effectively activate NK cells against AML targets in vitro and in vivo. This preclinical data has led to the establishment of a clinical trial at the University of Minnesota in patients with refractory AML, which is scheduled to begin in the third quarter of 2018. Although these previous studies have validated the use of TriKE as an effective strategy for utilizing NK cells in cancer immunotherapy, CD33 has limitations as a target antigen.

[0047] The high mortality and poor five-year survival rate (26%) of AML patients can be attributed to chemotherapy resistance and disease relapse. It is hypothesized that most chemotherapy-resistant leukemia stem cells (LSCs) that contribute to relapse do not express CD33. Furthermore, all hematopoietic stem cells and normal bone marrow cells express CD33, so targeting this antigen can lead to severe hematopoietic defects and on-target / off-tumor toxicity. To address these limitations, this article describes the development of TriKEs targeting CLEC12A or C-type lectin-like molecule 1 (CLL-1). CLEC12A is highly expressed on AML cells, and more than 70% of CD33-negative cells express CLEC12A. It has been recognized as a stem cell marker in AML and is selectively overexpressed in LSCs. In the regenerating bone marrow, CLEC12A is expressed by CD34+ / CD38- LSCs but not by normal CD34+ / CD38- hematopoietic stem cells, thereby minimizing off-target effects. C-type lectin domain family 12 member A is a human protein encoded by the CLEC12A gene. This gene encodes members of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share a common protein fold and have multiple functions, such as cell adhesion, intercellular signaling, glycoprotein turnover, and roles in inflammation and immune response. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternatively spliced ​​transcript variants of this gene have been described, but the full-length properties of some of these variants have not yet been determined. This gene is closely related to other CTL / CTLD superfamily members in the natural killer gene complex region on chromosome 12p13.

[0048] BiKE and TriKE compounds

[0049] Bispecific fusions have been performed that combine anti-human anti-CD16 scFv derived from human phage display library technology (McCall et al., 1999, Mol Immunol, 36: 433-445). NK cells mediate antibody-dependent cell-mediated cytotoxicity (ADCC) through the CD16 (FcγRIII) receptor. Signaling through the CD16 receptor induces calcium flux and phosphorylation of ITAMs, triggering the release of lytic granules and cytokines such as interferon (IFNγ) and tumor necrosis factor (TNFα). A bispecific molecule has been designed to trigger the CD16 receptor in combination with other targeting molecules (Gleason et al., Blood, 2014 (19): 3016-26), a so-called bispecific killer engager (BiKE). Because one scFv recognizes NK cells and the other scFv recognizes tumor antigens, BiKE can significantly enhance cytotoxic killing of a variety of human cancers. An exemplary BiKE targets CD33 and enhances NK cell responses against acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). MDS is a clonally heterogeneous stem cell disorder characterized by normocellular or hypercellular bone marrow (BM) with peripheral blood (PB) cytopenias and an increased risk of progression to AML.

[0050] NK cells respond to a variety of cytokines, including IL-15, which is involved in NK cell homeostasis, proliferation, survival, activation and / or development. For example, IL-15 can activate NK cells and can restore the functional defects of transplanted NK cells after hematopoietic stem cell transplantation (HSCT). IL-15 and IL-2 share several signaling components, including IL-2 / IL-15Rβ (CD122) and a common γ chain (CD132). Unlike IL-2, IL-15 does not stimulate Treg, allowing NK cell activation while bypassing the suppression of Treg on immune response. In addition to promoting NK cell homeostasis and proliferation, IL-15 can also rescue NK cell functional defects that may occur in the post-transplant environment. IL-15 can also stimulate CD8+T cell function, further enhancing its immunotherapy potential. In addition, according to preclinical studies, the toxicity profile of IL-15 may be more favorable than IL-2 at low doses. According to some embodiments, the compositions described herein can be used to activate NK cells and drive NK cell initiation, amplification and survival.

[0051] In one aspect, the present disclosure describes tri-specific killing junction (TriKE) molecules, which generally include one or more targeting domains (its targeting is for example tumor cells or virally infected cells) and one or more cytokine NK activation domains (such as IL-15, IL-12, IL-18, IL-21 or other NK cell enhancing cytokines, chemokines and / or activating molecules), wherein each domain is operably connected to other domains. As used herein, the term "operably connected" refers to a direct or indirect covalent connection. Therefore, two domains that are operably connected can be directly covalently coupled to each other. On the contrary, two domains that are operably connected can be connected by being covalently connected to a middle portion (for example, and flanking sequences) mutually. Two domains can be considered to be operably connected, for example, if they are separated by a third domain with or without one or more intermediate flanking sequences.

[0052] Exemplary BiKE and TriKE molecules or compounds are described in WO2017062604, the disclosure of which is incorporated herein by reference in its entirety.

[0053] In some embodiments, the present disclosure describes compounds comprising an NK engaging domain; an NK activation domain operably linked to the NK engaging domain; and a targeting domain that selectively binds to a target cell and is operably linked to the NK activation domain and the NK engaging domain, wherein the targeting domain selectively binds to a target molecule. For example, the target molecule can be expressed on the surface of a target cell. For example, the target cell can be a tumor cell. In some embodiments, the targeting domain selectively binds to CLEC12A.

[0054] As used herein, the term "selectively binds" or "selectively binds" with respect to the interaction of a binding molecule or domain (e.g., an antibody or engaging domain, activation domain, or targeting domain) described herein with its binding partner (e.g., an antigen or receptor) means that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope or amino acid sequence) on the binding partner. In other words, the binding molecule or domain preferentially binds to or recognizes the binding partner even if the binding partner is present in a mixture of other molecules. Binding can be mediated by covalent or non-covalent interactions, or a combination of both. The terms "selectively bind" or "selectively bind" and "specifically bind" or "specifically binds" can be used interchangeably.

[0055] The compounds described herein may or may not have a His tag. For example, a His tag allows for purification of proteins and can be used in research applications. The His tag can be located at the C-terminus or N-terminus of a compound or molecule described herein and can include a spacer at the N-terminus or C-terminus of the His tag. As an example, a His tag placed at the C-terminus of a compound or molecule described herein can include a spacer at the N-terminus of the His tag. As another example, a His tag placed at the N-terminus of a compound or molecule described herein can include a spacer at the C-terminus of the His tag. An exemplary His tag with a spacer is SEQ ID NO: 3. SEQ ID NO: 3 can be placed at the C-terminus of a compound described herein. One skilled in the art will appreciate that any number of His repeats can constitute a His tag and that any spacer sequence of any length or no spacer can be used.

[0056] In some embodiments, the TriKE compound or molecule that selectively binds to CLEC12A comprises the isolated amino acid sequence of SEQ ID NO: 1. In some embodiments, the TriKE compound or molecule that selectively binds to CLEC12A comprises the isolated amino acid sequence of SEQ ID NO: 2. In some embodiments, the targeting domain of the compounds described herein that selectively binds to CLEC12A comprises the isolated amino acid sequence of SEQ ID NO: 4.

[0057] Also described herein are nucleic acid sequences encoding the sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4. For example, SEQ ID NO: 1 can be encoded by SEQ ID NO: 5, SEQ ID NO: 2 can be encoded by SEQ ID NO: 6, and SEQ ID NO: 4 can be encoded by SEQ ID NO: 7. One skilled in the art will recognize that any functional variant of the nucleic acid molecules provided herein is encompassed by the present disclosure. A functional variant is a nucleic acid sequence that can be translated to provide an amino acid sequence homologous or identical to the amino acid sequence translated from the parent molecule.

[0058] NK engaging domain

[0059] The NK engaging domain may comprise any portion that binds to and / or activates NK cells and / or blocks any portion of the inhibition of NK cells. Exemplary NK cell engaging domains comprise portions that bind to, for example, CD16, CD16+CD2, CD16+DNAM, or CD16+NKp46. In some embodiments, the engaging domain comprises a portion that selectively binds to CD16. In some embodiments, the NK engaging domain activates NK cells. In some embodiments, the NK engaging domain blocks inhibition of NK cells.

[0060] In certain embodiments, the NK engagement domain may include an antibody that selectively binds to the surface components of NK cells. In other embodiments, the NK engagement domain may include a ligand or small molecule that selectively binds to the surface components of NK cells. As used herein, the term "selectively binds" refers to the ability to distinguish two or more alternatives, such as, for example, having any degree of differential affinity to a specific target. As used herein, "antibody" generally refers to an immunoglobulin or a fragment thereof, and therefore includes a monoclonal antibody, a fragment thereof (e.g., scFv, Fab, F (ab') 2, Fv or other modified forms), a combination of monoclonal antibodies and / or its fragments and / or a combination of polyclonal antibodies. Therefore, for simplicity, reference to the antibody that selectively binds to the surface components of NK cells includes any antibody fragment that shows the binding characteristics. Similarly, reference to the ligand that selectively binds to the surface components of NK cells includes any fragment of the ligand that shows the binding characteristics.

[0061] In certain embodiments, the NK engaging domain can selectively bind to a receptor at least partially located on the surface of an NK cell. In certain embodiments, the NK engaging domain can play a role in binding the function of NK cells, and thereby allow NK to spatially approach the target (described in more detail below) that the targeting domain selectively binds to. However, in certain embodiments, the NK engaging domain can selectively bind to the receptor that activates NK cells, and therefore also has an activation function. As described above, the activation of CD16 receptors can trigger antibody-dependent cell-mediated cytotoxicity. Therefore, in certain embodiments, the NK engaging domain can include at least a portion of an anti-CD16 receptor antibody that is effectively selectively bound to CD16 receptors. In other embodiments, the NK engaging sub-cell domain can interrupt the mechanism of suppressing NK cells. In such an embodiment, the NK engaging sub-domain can include, for example, anti-PD1 / PDL1, anti-NKG2A, anti-TIGIT, anti-KIR and / or any other inhibitory blocking domain.

[0062] Technicians can design NK engagement domains to have the desired degree of NK selectivity, and therefore have the desired immune engagement properties. For example, CD16 has been determined to be Fc receptor FcγRIIIa (CD16a) and FcγRIIIb (CD16b). These receptors bind to the Fc portion of IgG antibodies, which then activate NK cells for antibody-dependent cell-mediated cytotoxicity. Anti-CD16 antibodies selectively bind to NK cells, but can also bind to neutrophils. Anti-CD16a antibodies selectively bind to NK cells, but do not bind to neutrophils. TriKE embodiments comprising NK engagement domains (comprising anti-CD16a antibodies) can bind to NK cells, but do not bind to neutrophils. Therefore, in the case where the technician may want to engage NK cells but not engage neutrophils, the technician can design the NK engagement domain of TriKE to include anti-CD16a antibodies.

[0063] Although described herein in the context of various embodiments in which the NK engaging domain comprises an anti-CD16 receptor scFv, the NK engaging domain may comprise any antibody or other ligand that selectively binds to the CD16 receptor. In addition, the NK engaging domain may comprise an antibody or ligand that selectively binds to any NK cell receptor, such as, for example, cytotoxicity receptor 2B4, low affinity Fc receptor CD16, killer immunoglobulin-like receptor (KIR), CD2, NKG2A, TIGIT, NKG2C, LIR-1 and / or DNAM-1. In one embodiment, the composition of the present invention is a construct that is operably connected to NKG2C / IL-15 / CD33. It should be understood that the position of the portion may be changed based on an activity assay (e.g., CD33 / IL-15 / NKG2C).

[0064] In certain embodiments, the NK engaging domain comprises an antibody or its binding fragment or a nanobody. The antibody binding fragment can be scFv, F(ab)2 or Fab. In certain embodiments, the NK engaging domain comprises a nanobody. In certain embodiments, the NK cell engager can involve the use of a humanized CD16 engager derived from an animal nanobody. Although scFv has a heavy variable chain component and a light variable chain component connected by a joint, a nanobody is composed of a single monomer variable chain (i.e., a variable heavy chain or a variable light chain) that can specifically engage the target. Nanobodies can be derived from antibodies of any suitable animal (such as, for example, camelids (e.g., llamas or camels) or cartilaginous fish). Compared with larger antibody fragments, nanobodies can provide excellent physical stability, the ability to bind deep grooves, and increased yields.

[0065] In an exemplary embodiment, a nanobody-based NK engager molecule can involve a humanized CD16 nanobody derived from a published llama nanobody (GeneBank sequence EF561291; Behar et al., 2008, Protein Eng Des Sel, 21(1): 1-10), referred to as EF91. Llama EF91 was initially constructed as a BiKE containing CD19 to test the ability of this CD16 engager to drive NK cell activation. In a chromium release assay containing a Raji target, it showed function similar to rituximab-mediated killing. After confirming the function of the molecule, the CDRs were cloned into a humanized camelid scaffold (Vincke et al., 2009, J Biol Chem, 284(5): 3273-3284) to humanize the CD16 engager, now referred to as HuEF91. Binding of HuEF91 was equivalent to that observed using a standard CD16 scFv, indicating that incorporation of the rhea nanobody variable heavy chain into the humanized backbone did not hinder the specificity of the molecule. The use of HuEF91 as an NK engager in the TriKE molecules described herein may increase drug yield, increase stability, and / or increase NK cell-mediated ADCC potency.

[0066] Thus, according to some embodiments, the antibody or its binding fragment or nanobody is human or humanized.In some embodiments, the antibody or its binding fragment or nanobody is of Camelidae.

[0067] NK activation domain

[0068] NK activation domain can include an amino acid sequence that activates NK cells, promotes the maintenance of NK cells, or otherwise promotes NK cell activity. NK activation domain can be or can be derived from one or more cytokines that can activate and / or maintain NK cells. As used herein, the term "derived from" refers to an amino acid fragment sufficient to provide NK cell activation and / or maintain active cytokines (e.g., IL-15). In the embodiment comprising more than one NK activation domain, the NK activation domain can be provided in series or in any other combination. In addition, each NK activation domain based on a cytokine can include the complete amino acid sequence of a cytokine, or can be an amino acid fragment, regardless of the properties of the other NK activation domains included in the TriKE molecule. The exemplary cytokine that the NK activation domain can be based on includes, for example, IL-15, IL-18, IL-12, and IL-21. Therefore, although this article describes in detail in the context of the exemplary model embodiment in which the NK activation domain is derived from IL-15, TriKE can be designed using NK activation domains, and the NK activation domains are or are derived from any suitable cytokine.

[0069] For the sake of brevity, in this specification, references to NK activation domains by identifying the cytokine on which the NK activation domain is based include the complete amino acid sequence of the cytokine, any suitable amino acid fragment of the cytokine, and / or a modified version of the cytokine comprising one or more amino acid substitutions. Thus, reference to an "IL-15" NK activation domain includes an NK activation domain comprising the complete amino acid sequence of IL-15, an NK activation domain comprising a fragment of IL-15, or an NK activation domain comprising amino acid substitutions compared to the wild-type IL-15 amino acid sequence, such as, for example, IL-15N72D or IL-15N72A.

[0070] The use of the IL-15 NK activation domain in TriKE can provide sustained NK cell activity (as demonstrated in a mouse model that showed a significant increase in human NK cells and a reduction in cancer), even after three weeks. NK cells are activated in mice to produce a range of anti-cancer factors and cytokines. In addition, the IL-15 NK activation domain can change the chemical properties of these molecules, making them more easily refolded and / or recovered in higher yields, making TriKE molecules more suitable for clinical scale-up.

[0071] Thus, in some embodiments, the NK activation domain comprises a cytokine or a functional fragment thereof. In some embodiments, the activation domain comprises IL-15 or a functional fragment thereof. In some embodiments, the IL-15 is wild-type IL-15. In some embodiments, the IL-15 is human. In some embodiments, the IL-15 is wild-type human IL-15. In some embodiments, the IL-15 comprises the amino acid sequence of SEQ ID NO: 9 or a functional variant thereof. In some embodiments, the functional variant of IL-15 comprises an N72D or N72A amino acid substitution compared to SEQ ID NO: 9.

[0072] As used herein, the term "functional variant" refers to a molecule (comprising binding molecules) that, for example, comprises nucleotides and / or amino acid sequences that are changed by one or more nucleotides and / or amino acids compared to the nucleotides and / or amino acid sequences of the parental molecule. For binding molecules, functional variants can still compete with the parental binding molecules to bind to the binding partner. In other words, the modification in the amino acid and / or nucleotide sequence of the parental binding molecules will not significantly affect or change the binding properties of the binding molecules encoded by the nucleotide sequence or containing the amino acid sequence, i.e., the binding molecules can still recognize and combine their targets. Functional variants can have conservative sequence modifications, comprising nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art (such as site-directed mutagenesis and random PCR-mediated mutagenesis).

[0073] Functional variants can also include, but are not limited to, derivatives that are substantially similar in primary structural sequence but contain in vitro or in vivo chemical and / or biochemical modifications not found in the parent binding molecule, for example. Such modifications include, inter alia, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI-anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, ubiquitination, etc.

[0074] Targeting domain and target

[0075] The targeting domain can comprise any moiety that selectively binds to an intended target, such as, for example, a tumor cell, a target in a cancer stroma, a target on a suppressor cell such as a myeloid-derived suppressor cell that is CD33+, or a target on a virally infected cell. Thus, the targeting domain can comprise, for example, an anti-tumor antibody such as rituximab (anti-CD20), afutuzumab (anti-CD20), trastuzumab (anti-HER2 / neu), pertuzumab (anti-HER2 / neu), labetuzumab (anti-CEA), adelimumab (anti-EpCAM), pocitabine (anti-EpCAM), edrecolomab (anti-EpCAM), acitumomab (anti-CEA), bevacizumab (anti-VEGF-A), cetuximab (anti-EGFR), nimotuzumab (anti-EGFR), panitumumab (anti-EGFR), zalutumumab (anti-EGFR), gemtuzumab tuzumab (anti-CD33), lintuzumab (anti-CD33), edamasumab (anti-integrin α v Any tumor marker can be targeted. In some embodiments, the targeting domain or the targeted tumor-associated antigen may comprise CD133, CD20, HER2, CEA, EpCAM, VEGF-A, EGFR, CD33, integrin αVβ3, CD51, CD152, CD125, CTAA16.88, MUC1, CD19, CD22, CD38, mesothelin, ROR1, CSPG4, SS1 or IGFR1, NKG2 family members, such as but not limited to 2A, 2B, 2C, etc., BCMA, APRIL, B7H3 and PSMA.

[0076] In some embodiments, the target cell is a tumor cell. In some embodiments, the tumor cell is CD33+. In some embodiments, the tumor cell is CD33-. In some embodiments, the tumor cell is a hematopoietic cancer cell. In some embodiments, the tumor cell is a leukemia cell. In some embodiments, the leukemia cell is an acute myeloid leukemia (AML) cell. In other embodiments, the targeting domain can selectively bind to a target on a cell infected by a virus, such as, for example, EBV, HBV, HCV and / or HPV. In some embodiments, the viral target is a tumor marker or tumor antigen. Any viral tumor marker or viral or non-viral tumor antigen can be targeted.

[0077] As mentioned above, the targeting domain portion can comprise an antibody or an antibody binding fragment or a nanobody. The antibody binding fragment can include scFv, F(ab)2 or Fab.

[0078] In certain specific embodiments, the targeting domain may comprise an anti-CLEC12A antibody. In other specific embodiments, a second targeting domain may be included. The second targeting domain may comprise a portion that can bind to any of the above targets. In some embodiments, the second targeting domain can selectively bind to CD33.

[0079] In some embodiments, the compounds described herein comprise an NK engaging domain having a portion that selectively binds to CD16, an activation domain having IL-15, and a targeting domain that selectively binds to CLEC12A. The terms "CLEC12ATrike," "1615CLEC12ATriKe," and "CD16-IL15-CLEC12ATriKE" are used interchangeably to refer to TriKEs that target CLEC12A, unless the context clearly indicates otherwise.

[0080] Flanking sequences

[0081] In some embodiments, the compounds described herein may further include flanking sequences or joint sequences that can connect two of the above-mentioned domains. Unless the context clearly indicates otherwise, the terms "flanking sequence" and "joint sequence" can be used interchangeably. In some embodiments, the presence of flanking sequences can further increase NK cell activation. Any amino acid sequence can be a flanking sequence or joint sequence. An exemplary flanking sequence includes 20 amino acids of SEQ ID NO:13. Another exemplary flanking sequence includes seven amino acids of SEQ ID NO:14. Some other exemplary flanking sequences include SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:15. As another example, any number of repetitions of an amino acid sequence can be a flanking sequence or joint sequence. For example, any number of repetitions of the sequence of SEQ ID NO:15 can be a flanking sequence or joint sequence. Sequence repetitions can be complete or partial, and complete or partial repetitions can be at the beginning (i.e., at the N-terminus) or end (i.e., at the C-terminus) of a flanking sequence or joint sequence. Flanking sequences can be in any direction.

[0082] Certain embodiments (e.g., 1615CLEC12ATriKE without a His tag, SEQ ID NO: 1, or 1615CLEC12ATriKE with a His tag, SEQ ID NO: 2) may include more than one flanking sequence. As an example, SEQ ID NO: 1 and / or SEQ ID NO: 2 include flanking sequences of SEQ ID NO: 11 to connect the NK engagement domain (e.g., anti-CD16 receptor scFv) to the NK activation domain (e.g., IL-15). SEQ ID NO: 1 and / or SEQ ID NO: 2 further include flanking sequences of SEQ ID NO: 12 to connect the NK activation domain to the targeting domain (e.g., anti-CLEC12A scFv). The flanking sequences of the domains of the connecting molecules may be the same or different. As an example, the same or different flanking sequences can link an NK engaging domain (e.g., an anti-CD16 receptor scFv) to an NK activation domain (e.g., IL-15), and link an NK activation domain to a targeting domain (e.g., an anti-CLEC12A scFv). In some embodiments, constructs lacking flanking sequences exhibit reduced activity compared to constructs with flanking sequences.

[0083] In some embodiments, the compounds described herein comprise at least one flanking sequence connecting the two domains. In some embodiments, the compounds described herein further comprise a second flanking sequence connecting the two connected domains to a third domain. In some embodiments, the flanking sequences are identical. In some embodiments, the flanking sequences are different.

[0084] In some embodiments, the flanking sequence flanks the NK activation domain. In some embodiments, the first flanking sequence is the C-terminus of the NK engaging domain. In some embodiments, the second flanking sequence is the N-terminus of the anti-CLEC12A targeting domain. In some embodiments, the first flanking sequence is the C-terminus of the NK engaging domain and the second flanking sequence is the N-terminus of the anti-CLEC12A targeting domain.

[0085] preparation

[0086] The compounds described herein can be formulated with a pharmaceutically acceptable carrier. As used herein, a "carrier" comprises any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, etc. The use of such media and / or agents for pharmaceutically active substances is well known in the art. Except for the case where any conventional media or agents are incompatible with the active ingredient, their use in therapeutic compositions is also contemplated. Supplementary active ingredients can also be incorporated into the composition. As used herein, "pharmaceutically acceptable" refers to a non-biologically or otherwise undesirable material, i.e., the material can be administered to an individual together with the TriKE molecule without causing any undesirable biological effect or interacting in a harmful manner with any other component of the pharmaceutical composition containing the material.

[0087] Thus, the TriKE molecules can be formulated into pharmaceutical compositions. The pharmaceutical compositions can be formulated into various forms suitable for preferred routes of administration. Thus, the compositions can be administered by known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.) or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transdermal, rectal, etc.). The pharmaceutical compositions can be administered to mucosal surfaces, such as by administration to, for example, the nose or respiratory mucosa (e.g., by sprays or aerosols). The compositions can also be administered by sustained release or delayed release.

[0088] Thus, the TriKE molecule can be provided in any suitable form, including but not limited to a solution, suspension, emulsion, spray, aerosol, or any form of mixture. The composition can be delivered in a formulation with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation can be delivered in a conventional topical dosage form such as, for example, a cream, ointment, aerosol formulation, non-aerosol spray, gel, lotion, etc. The formulation can further comprise one or more additives, including, for example, adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, humectants, thickeners, etc.

[0089] The formulations can be conveniently provided in unit dosage form and can be prepared by methods well known in the pharmaceutical art. Methods for preparing compositions with pharmaceutically acceptable carriers comprise the step of associating the TriKE molecule with a carrier which constitutes one or more auxiliary ingredients. In general, the formulations can be prepared by uniformly and / or intimately associating the active molecule with a liquid carrier, a finely divided solid carrier, or both, and then, if desired, shaping the product into the desired formulation.

[0090] Treatment

[0091] In some embodiments, provided herein are methods comprising administering to a subject a compound or molecule described herein in an amount effective to induce NK-mediated killing of a target cell. Any cell can be a target cell. In some embodiments, the target cell is a cancer cell. The methods described herein may comprise administering to a subject a TriKE molecule in an amount effective to induce NK-mediated killing of a target cell in the subject. In some embodiments, the TriKE molecule is administered to treat a disease or condition in the subject.

[0092] As used herein, "treat," or variations thereof, refers to alleviating, limiting the progression, ameliorating, or resolving to any extent the symptoms or signs associated with a condition. As used herein, "improvement" refers to any reduction in the extent, severity, frequency, and / or likelihood of symptoms or clinical signs characteristic of a particular condition; "symptoms" refers to any subjective evidence of a disease or condition in a patient; and "signs" or "clinical signs" refers to objective physical findings associated with a particular condition that can be observed by a person other than the subject or patient.

[0093] As used herein, the term "subject" refers to any individual or patient to whom the methods disclosed herein are performed. The term "subject" can be used interchangeably with the term "individual" or "patient." A "subject" can be any animal, such as, for example, a mammal (e.g., a dog, cat, horse, cow, sheep, goat, monkey, etc.). In certain embodiments, the subject can be a human.

[0094] "Treatment" can be therapeutic or prophylactic. "Therapeutic" and its variants refer to treatment that improves one or more existing symptoms or clinical signs associated with a condition. "Prophylactic" and its variants refer to treatment that limits the development and / or appearance of symptoms or clinical signs of a condition to any extent. Generally speaking, "therapeutic" treatment begins after the condition manifests in the subject, while "prophylactic" treatment begins before the condition manifests in the subject. Therefore, in certain embodiments, the method may include prophylactic treatment of a subject at risk of developing the condition. "At risk" means that the subject may or may not actually have the risk. Thus, for example, a subject "at risk" for developing a particular condition is a subject who has one or more markers that increase the risk of having or developing the particular condition, regardless of whether the subject exhibits any symptoms or clinical signs of having or developing the condition, compared to an individual who lacks one or more markers. Exemplary markers of a condition may include, for example, genetic predisposition, ancestry, age, sex, geographic location, lifestyle, or medical history. Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[0095] In other embodiments, provided herein is a method for stimulating the expansion of NK cells in vivo, comprising administering to a subject a compound or molecule as described herein in an amount that effectively stimulates the expansion of NK cells in the subject. In certain embodiments, TriKE molecules are administered to treat a disease or condition in the subject. Using TriKE molecules as part of an in vivo treatment can make NK cells antigen-specific, while performing co-stimulation, survival enhancement, and expansion, which may be antigen-specific. In other cases, TriKE can be used as an adjuvant for NK cell adoptive transfer therapy in vitro.

[0096] In other embodiments, provided herein are methods for treating cancer comprising administering to a subject a compound or molecule described herein that is effective for treating cancer. In some embodiments, the cancer is prostate cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, melanoma, kidney cancer, renal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, or a hematopoietic cancer. In some embodiments, the hematopoietic cancer is myelodysplastic syndrome (MDS). In some embodiments, the hematopoietic cancer is a lymphoma. In some embodiments, the hematopoietic cancer is a leukemia. In some embodiments, the hematopoietic cancer is acute myeloid leukemia (AML).

[0097] As used herein, the term " myeloid leukemia " refers to the leukemia characterized by the abnormal increase of the number of granulocytes, bone marrow cells and myeloblasts in the proliferation and circulating blood of myeloid tissue. The term is synonymous with term myeloid leukemia, myeloid leukemia, myeloid leukemia and granulocytic leukemia. The term " myeloid leukemia " can especially represent acute and chronic myeloid leukemia (AML and CML), acute promyelocytic leukemia (APL), chronic myelomonocytic leukemia (" CMML "), myelodysplastic syndrome and juvenile myelomonocytic leukemia, which relates to the myeloid components (for example, leukocytes, erythrocytes and megakaryocytes) of bone marrow, and includes all subtypes defined by morphology, histochemistry and immunological techniques well known to those skilled in the art. The subtype of AML includes FAB-M0, FAB-M1, FAB-M2, FAB-M3, FAB-M4, FAB-M5, FAB-M6 and FAB-M7 classified according to FAB.

[0098] As used herein, the term "myelodysplastic syndrome" encompasses a heterogeneous group of closely related clonal hematopoietic disorders originating from early hematoblasts in the bone marrow. All disorders are characterized by a bone marrow with impaired morphology and maturity (myelopoiesis) and peripheral cytopenias, which are caused by ineffective hematopoiesis. In other words, mature blood cells typically die in the bone marrow before they reach full maturity and enter the blood, which is the reason for the low blood cell concentration. In patients with myelodysplastic syndrome, there may also be an accumulation of very immature bone marrow cells (called leukemic blasts).

[0099] The amount of a TriKE molecule administered can vary depending on various factors, including, but not limited to, the specific TriKE molecule used, the weight, physical condition, and / or age of the subject, and / or the route of administration. Thus, the absolute weight of a TriKE molecule contained in a given unit dosage form can vary widely and depend on factors such as the species, age, weight, and physical condition of the subject, and / or the method of administration. Thus, it is not practical to generally specify an amount that will constitute an effective amount of a TriKE molecule for all possible applications. However, one of ordinary skill in the art can readily determine an appropriate amount with due consideration of these factors.

[0100] In some embodiments, the method can comprise administering a sufficient amount of the TriKE molecule to provide a dose of, for example, about 100 ng / kg to about 50 mg / kg to the subject, although in some embodiments the method can be performed by administering a dose of the TriKE molecule outside this range. In some of these embodiments, the method comprises administering a sufficient amount of the TriKE molecule to provide a dose of about 10 μg / kg to about 5 mg / kg to the subject, for example, about 100 μg / kg to about 1 mg / kg.

[0101] Alternatively, the dose can be calculated using the actual body weight obtained just before the start of the treatment course. For doses calculated in this manner, body surface area (m2) is calculated before the start of the treatment course using the Dubois method: m2 = (wt kg 0.425 × height cm 0.725) × 0.007184.

[0102] In some embodiments, the methods can comprise administering a sufficient amount of the TriKE molecule to provide a dose of, for example, about 0.01 mg / m2 to about 10 mg / m2.

[0103] In some embodiments, the TriKE molecule can be administered, for example, from a single dose to multiple doses per week, although in some embodiments the method can be performed by administering the TriKE molecule at a frequency outside of this range. In certain embodiments, the TriKE molecule can be administered from about once per month to about five times per week.

[0104] In some embodiments, the method further comprises administering one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered before, after, and / or simultaneously with the administration of the TriKE molecule. The TriKE molecule and the additional therapeutic agent can be co-administered. As used herein, "co-administration" refers to two or more components administered in combination such that the therapeutic or preventive effect of the combined administration is greater than the therapeutic or preventive effect of either component administered alone. The two components can be co-administered simultaneously or sequentially. The components co-administered simultaneously can be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes situations in which the components are administered such that each component can be present at the treatment site simultaneously. Alternatively, sequential co-administration of two components can include situations in which at least one component has been cleared from the treatment site, but at least one cellular effect of administering the component (e.g., cytokine production, activation of a specific cell population, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Thus, in some cases, a co-administered combination can include components that have never been present in a chemical mixture with each other. In other embodiments, the TriKE molecule and the additional therapeutic agent can be administered as part of a mixture or admixture. In some aspects, administration of a TriKE molecule can allow for the effectiveness of lower doses of the other therapeutic modality when compared to administration of the one or more other therapeutic agents alone, thereby reducing the likelihood, severity, and / or extent of toxicities observed when higher doses of the one or more other therapeutic agents are administered.

[0105] Exemplary additional therapeutic agents include, for example, hexamethonium, amsacrine, L-asparaginase, L-asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytosine, cytarabine, dacarbazine, actinomycin D, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, Fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, and vinorelbine.

[0106] Thus, in some embodiments, the methods of treating cancer provided herein further comprise administering a compound, molecule, composition or formulation as described herein before, concurrently with, or after chemotherapy, surgical resection of a tumor, or radiation therapy. Chemotherapy can comprise, for example, hexamethonium, amsacrine, L-asparaginase, L-asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytosine, cytarabine, dacarbazine, actinomycin D, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, formox, statins, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, thioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, and vinorelbine.

[0107] In some embodiments, the methods provided herein can comprise administering sufficient TriKE molecules as described herein and administering at least one additional therapeutic agent, wherein administration of the TriKE molecules and at least one additional therapeutic agent demonstrates therapeutic synergy. In some aspects of the methods of the invention, a measure of response to treatment observed after administration of a TriKE molecule as described herein and an additional therapeutic agent is improved compared to the same measure of response to treatment observed after administration of the TriKE molecule or the additional therapeutic agent alone. In some embodiments, the additional therapeutic agent can comprise an additional agent that targets EpCAM, including, for example, an EpCAM-specific monoclonal antibody, such as, for example, catumaxomab, a monoclonal hybrid antibody that targets EpCAM and CD3.

[0108] As used herein, the term "and / or" refers to one or all of the listed elements or any combination of two or more of the listed elements; the terms "comprises," "comprising," and variations thereof should be interpreted as open-ended, i.e., additional elements or steps are optional and may or may not be present.

[0109] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise indicated, "a," "an," "the," and "at least one" are used interchangeably and may mean one or more than one. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein, as will become apparent to those skilled in the art upon reviewing this disclosure and the like.

[0110] As used herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (eg, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0111] As used herein, "about" when referring to a measurable value such as an amount, a period of time, etc., is meant to encompass variations of ±20% or ±10% or ±5% or even ±1% from the specified value, as such variations are suitable for the disclosed methods or for performing the disclosed methods.

[0112] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0113] As used herein, the term "protein" refers to any polymer chain of amino acids. The terms "peptide" and "polypeptide" can be used interchangeably with the term "protein" and also refer to a polymer chain of amino acids. The term "protein" includes natural or artificial proteins, protein fragments, and polypeptide analogs of protein sequences. Proteins can be monomeric or polymeric. The term "protein" includes fragments and variants thereof (including fragments of variants), unless the context otherwise contradicts.

[0114] As used herein, the term "nucleic acid" refers to any deoxyribonucleic acid (DNA) molecule, ribonucleic acid (RNA) molecule, or nucleic acid analog. The DNA or RNA molecule can be double-stranded or single-stranded and can be of any size. Exemplary nucleic acids include, but are not limited to, chromosomal DNA, plasmid DNA, cDNA, cell-free DNA (cfDNA), mRNA, tRNA, rRNA, siRNA, microRNA (miRNA or miR), hnRNA. Exemplary nucleic acid analogs include peptide nucleic acids, morpholino and locked nucleic acids, glycol nucleic acids, and threose nucleic acids.

[0115] In the foregoing description, specific embodiments may be described independently for clarity. Unless otherwise explicitly specified that features of a specific embodiment are incompatible with features of another embodiment, some embodiments may include a combination of compatible features described herein in conjunction with one or more embodiments.

[0116] For any method comprising discrete steps disclosed herein, these steps may be performed in any feasible order. In addition, any combination of two or more steps may be performed simultaneously, where appropriate.

[0117] The present invention is illustrated by the following examples. It is to be understood that the specific examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as described herein.

[0118] Example

[0119] Example 1

[0120] This example describes the development of a CD16-IL15-CLEC12ATriKE.

[0121] 1615CLEC12ATriKE was developed in a mammalian cell system to ensure the presence of appropriate post-translational modifications. Specific binding of the TriKE to HL 60 and THP 1 target cells expressing CLEC12A was confirmed compared to Raji cells that do not express CLEC12A. Treatment of peripheral blood mononuclear cells (PBMCs) with 1615CLEC12ATriKE drove a significant increase in NK cell-specific proliferation within 7 days compared to treatment with CLEC12AscFv or IL-15 alone, as measured by CellTrace dilution (69.7±6.7% / 11.9±2.5% / 38.4±7.3%) ( Figure 1A ). To measure NK cell killing, an IncuCyte scaling assay was performed. Here, HL-60 target cells were labeled with a caspase 3 / 7 reagent, where a color change indicates target cell death. As measured by the number of viable target cells at the end of the 48-hour assay, 1615CLEC12A TriKE was able to induce more target cell killing than CLEC12A scFv or IL-15 (53.9±1.9% / 103.3±3.4% / 71.1±1.4%). Compared to treatment with CLEC12A scFv or IL-15 alone, 1615CLEC12A TriKE induced an increase in NK cell degranulation, as measured by CD107a expression against HL-60 AML tumor targets in a 4-hour functional assay (62.3±1.1% / 19.4±3.8% / 27.5±4.9%). In this assay, there was also an increase in cytokine production, as measured by IFNg and TNFa (16.7±4.2% / 2.3±1.5% / 4.7±1.9% and 18.0±5.1% / 2.5±1.7% / 4.6±2.5%), respectively ( Figure 1B). Similar enhanced functional responses were observed with THP-1 AML tumor targets. In these functional assays, treatment with 1615CLEC12ATriKE resulted in less background activation compared to CD33 TriKE, indicating less off-target effects on PBMCs. To confirm the clinical relevance of this molecule, the efficacy of 1615CLEC12ATriKE against primary AML targets was tested. AML blasts were identified as SSC low, CD45 intermediate and CD34 high cells. Of the 9 AML samples tested, 7 expressed high levels of CD33 (70.4±6.3%) and CLEC 12a (78.1±5.2%). In functional assays performed on these samples, 1615CLEC12A TriKE was able to induce higher expression of CD107a and IFNg as well as enhanced killing of tumor targets as determined by live / dead staining compared to CLEC12AscFv or IL-15 ( Figure 1C ). In these assays, 1615CLEC12ATriKE demonstrated comparable potency to CD33 TriKE. These data demonstrate that 1615CLEC12ATriKE drives NK cell-specific proliferation, degranulation, cytokine secretion, and tumor target killing in vitro. In addition to AML, CLEC12A is also expressed on cancer cells and LSCs from patients with myelodysplastic syndrome (MDS). These findings highlight the clinical potential of 1615CLEC12ATriKE, alone or in combination with CD33 TriKE, for the treatment of MDS and AML.

[0122] Example 2

[0123] This example describes the expression of CD33 and CLEC12A on AML cells.

[0124] Most of all deaths from hematopoietic malignancies are caused by acute myeloid leukemia (AML), which has a poor five-year survival rate of 26%, highlighting the need for new therapies. The most common antigen used to target AML cells is CD33. However, there are many limitations to the development of anti-CD33 therapies. For example, not all cancer cells express CD33, including those in patients with refractory AML. In addition, all cells of the myeloid lineage and some cells of the lymphoid lineage, such as activated NK cells and T cells, express CD33, leading to off-target toxicity. In addition, cancer stem cells, which are thought to contribute to relapse, do not express CD33.

[0125] A new antigen called C-type lectin-like molecule 1 (CLL-1), or CLEC12A, aims to address these limitations.

[0126] Figure 2Shown are the percentages of CD33 and CLEC12A surface expression measured by flow cytometry analysis of primary AML samples from 10 patients. CLEC12A is highly expressed on AML cells. Approximately 70% of CD33-negative cells express CLEC12A. CLEC12A expression is restricted to a subset of myeloid cells, limiting off-target toxicity. CLEC12A is present on leukemic stem cells but not on hematopoietic stem cells.

[0127] These data establish CLEC12A as a surface marker on primary AML cells that express and lack CD33 expression.Thus, according to some embodiments, CLEC12A can be targeted by trispecific killer cell engager (TriKE) molecules on AML and other cells that express or lack CD33.

[0128] Example 3

[0129] This example describes a trispecific killer-engager (TriKE) molecule that targets CLEC12A.

[0130] To target cancer cells using natural killer (NK) cells, a trispecific killer engager (TriKE) molecule was developed that contains an anti-CD16 heavy chain antibody that activates NK cells, an IL-15 molecule that drives NK cell priming, expansion, and survival, and an anti-CLEC12A single-chain variable fragment (scFv) that engages the cancer target. Figure 3A -B shows a schematic diagram of the CD16-IL15-CLEC12A TriKE and its mechanism of action.

[0131] TriKE contains a llama anti-CD16 V HH The CDRs were integrated into the humanized V HH The anti-CD16 heavy chain antibody constructed in the main chain ( Figure 3A ). This is associated with a wild-type IL-15 molecule that is linked to a scFv from an anti-CLEC12A antibody. TriKE (SEQ ID NO.: 1) is produced in a mammalian system containing Expi-293 cells and contains a His tag for purification of the molecule. According to some embodiments, the TriKE molecule may lack a His tag. TriKE molecules lacking a His tag may be suitable for clinical applications, although TriKE containing a His tag may also be used. TriKE forms an immune synapse between CLEC12A+ tumor cells and NK cells, promoting the release of cytotoxic granules and the secretion of cytokines that kill target cells ( Figure 3B ).

[0132] In addition to the scFv from the anti-CLEC12A antibody (SEQ ID NO.: 4; corresponding to SC02-357 of U.S. Patent No. 7,741,443) described above as an illustrative example, the TriKE targeting domain can also include any sequence capable of targeting or binding to CLEC12A, such as scFvs SC02-378 and SC02-161, and any derivatives of scFvs SC02-357, SC02-378, and SC02-161. scFvs SC02-357, SC02-378, and SC02-161 are described in U.S. Patent No. 7,741,443, the disclosure of which is incorporated herein in its entirety, particularly with respect to the sequences of scFvs SC02-357, SC02-378, and SC02-161.

[0133] Example 4

[0134] This example describes the validation of CD16-IL15-CLEC12A TriKE binding to target cells.

[0135] CLEC12A+HL-60 and CLEC12A-Raji targets were incubated with equimolar concentrations of 1615CLEC12A TriKE or scFv. Binding was assessed using an anti-His antibody that binds to the His tag on the TriKE or scFv. A secondary streptavidin antibody was used for detection by flow cytometry. Figure 4 The data in show that 1615CLEC12A TriKE binds to the HL-60 target but not to the Raji target.

[0136] Using a target against CD16 ( Figure 14A ), IL15 receptor α ( Figure 14B ) and CLEC12A extracellular domain (ED; Figure 14C The binding of the different components of 1615CLEC12A TriKE was tested by ELISA based on ELISA kits. Unless otherwise stated, TriKE was tested in a 3-fold serial dilution from 900 nM to 0.4 nM, and the highest binding was at 30 nM, which was used for subsequent experiments.

[0137] These data indicate that each component of 1615CLEC12ATriKE binds to its respective target molecule and that CD16-IL15-CLEC12ATriKE specifically binds to targets expressing CLEC12A.

[0138] Example 5

[0139] This example describes NK cell proliferation induced by the CD16-IL15-CLEC12A TriKE.

[0140] PBMCs were labeled with Cell Trace and incubated with equimolar concentrations of IL-15, CLEC12A scFv, or CLEC12ATriKE for 7 days ( Figure 5A NK cell populations were assessed by estimating the dilution of Cell Trace dye in the CD56+CD3- population using flow cytometry ( Figure 5B The percentage of proliferating NK cells was calculated using a FlowJo Analyzer. Statistics reflect significant differences between groups as calculated using one-way AVONA, *P < 0.05, **P < 0.005, N = 10. A higher percentage of proliferating NK cells was observed in the presence of CLEC12A TriKE compared to IL-15 or CLEC12AscFv.

[0141] In other experiments, PBMCs were isolated from fresh healthy donor samples (n=6), labeled with CellTrace Violet, and incubated for 7 days with 30 nM 1615CLEC12A TriKE or control treatment as described above. After the incubation period, cells were harvested and NK cell (CD3-, CD56+) proliferation was assessed by flow cytometry.

[0142] Aggregated data ( Figure 9A ) and representative histograms ( Figure 9B ) shows NK cell proliferation (by CellTrace dilution) in different treatment groups. Figure 9C The mixed NK cell counts during harvest are shown (at a constant speed of 45 seconds). One-way analysis of variance (ANOVA) with repeated measures was used to calculate the differences compared to the 1615CLEC12A group. Error bars represent the + / - standard error of the mean. Statistical significance was determined as **P<0.005, ****P<0.0001. Compared to no treatment, IL-15 treatment, or CLEC12AscFv treatment, significantly higher percentages of proliferated NK cells were observed after treatment with CLEC12A TriKE.

[0143] These data indicate that 1615CLEC12A TriKE induced potent NK cell proliferation.

[0144] Example 6

[0145] This example describes the functional validation of the CD16-IL15-CLEC12ATriKE.

[0146] PBMCs were incubated with CLEC12A+HL-60 and THP1 cells at an effector to target ratio of 2:1 in the presence of equimolar concentrations of IL-15, CLEC12A scFv, or CLEC12ATriKE. + CD3 - Surface CD107a was assessed on NK cells to estimate degranulation ( Figure 6A ), intracellular IFNg( Figure 6B ) and TNFa to estimate the production of inflammatory cytokines ( Figure 6C ). Statistics comparing treatment with CLEC12A TriKE to treatment with IL-15 or CLEC12A scFv control reflect significant differences between groups as calculated using one-way ANOVA, **P < 0.005, N = 6. A higher percentage of CD107a surface staining and a higher percentage of IFNg and TNFa intracellular staining on NK cells were observed in the presence of CLEC12A TriKE compared to no treatment, treatment with IL-15, or treatment with CLEC12A scFv on HL60 and THP1 target cells.

[0147] In other experiments, frozen PBMCs from healthy donors (n=6) were incubated with the indicated treatments (30 nM) to estimate CD107a expression as a function of degranulation ( Figure 10A ), intracellular IFNg production ( Figure 10B ) or intracellular TNFα expression in NK cells (CD3-, CD56+; Figure 10C ) marker. Cells were estimated using PBMCs alone or in the presence of THP1 and HL-60 targets at an effector / target ratio of 2:1. CD69 expression was used to estimate activation of NK cells (CD3-, CD56+) in PBMCs alone or in the presence of THP1 and HL-60 targets at an effector / target ratio of 2:1 in a 4-hour assay. Figure 10D ). Differences for the 1615CLEC12A group were calculated using one-way analysis of variance (ANOVA) with repeated measures. Error bars represent + / - standard error of the mean. Statistical significance was determined as *P, .05, **P, .01, ***P, .001, and ****P, .0001.

[0148] Greater NK cell activation was observed when incubated with CLEC12A TriKE compared to no treatment, IL-15 treatment, or scFv treatment of THP1 and HL60 target cells, as demonstrated by increased staining for CD107a, IFNg, TNFa, and CD69 ( Figure 10A -D).

[0149] These data indicate that 1615CLEC12ATriKE induces degranulation and cytokine production against AML target cells, including THP1 and HL-60 targets.

[0150] Example 7

[0151] This example describes CD16-IL15-CLEC12A TriKE-induced killing of AML targets.

[0152] In the presence of equimolar concentrations of IL-15, CLEC12AscFv or CLEC12ATriKE, the enriched NK cells were mixed with CLEC12A+HL-60 ( Figure 7A ) and THP1( Figure 7B ) targets were incubated with an effector to target ratio of 2: 1. Target cells were labeled with Cell TraceFar Red dye and caspase 3 / 7 green apoptosis assay reagent (Essen Biosciences). Killing was assessed using an Incucyte scaler and analyzed by normalizing cell number to the initial number of target cells. Figure 7A -B depicts the following situations (from top): (i) no treatment (first from the top); (ii) treatment with CLEC12AscFv (second from the top); (iii) treatment with IL-15 (third from the top / second from the bottom); (iv) treatment with CLEC12ATriKE (fourth from the top / bottom). The percentage of live target cells was lowest after treatment with CLEC12ATriKE.

[0153] Induction of 1615CLEC12ATriKE-mediated target cell killing was assessed in a real-time imaging assay. Enriched NK cells (CD3-, CD56+) were incubated with CellTrace Far Red-labeled THP-1 cells at a 2:1 effector to target ratio in an IncuCyte S3 imager with the treatment (30 nM) for 48 hours. Dead THP-1 cells were assayed using a caspase 3 / 7 reagent. Figure 11A Quantification of the percentage of viable THP-1 tumor targets (CellTrace FarRed / Caspases 3 / 7) normalized to the individual targets at the 0 hour time point is shown. Readings were taken every 30 minutes over 48 hours. Representative of three separate experiments. The first from the top corresponds to no treatment, the second from the top corresponds to treatment with CLEC12AscFv, the third from the top corresponds to treatment with IL-15, and the fourth from the top corresponds to treatment with CLEC12ATriKE. The lowest percentage of viable THP-1 cells was observed in the presence of CLEC12ATriKE.

[0154] Figure 11B Representative images after 0, 18, and 36 hours (original magnification 34: 2.82 mm / pixel) show THP-1 cells (larger cells) and NK cells (smaller cells). After 0 hour, there were almost no dead cells for all indicated treatment conditions. After 18 and 36 hours, there were almost no dead THP-1 cell clusters under all no treatment and CLEC12A scFv conditions, with some dying THP-1 cell clusters for IL-15 treatment and more dying cell clusters in the case of CLEC12A TriKE treatment.

[0155] Figure 11C Quantification of the percentage of live THP-1 tumor targets at different effector-to-target ratios (1:1, 2:1, and 5:1) is shown. The first, second, and third from the top correspond to NK alone, the fourth from the top corresponds to 1:1 NK+CLEC12A TriKE, the fifth from the top corresponds to 2:1 NK+CLEC12ATriKE, and the sixth from the top corresponds to 5:1 NK+CLEC12ATriKE. Compared to NK cells alone, a lower percentage of live THP-1 cells was observed for all effector-to-target ratios, with a 5:1 effector-to-target ratio resulting in the lowest percentage of live THP-1 cells, but this decrease in THP-1 cell viability was greatest following treatment with CLEC12ATriKE.

[0156] In other experiments, enriched NK cells were incubated with CellTrace Far Red-labeled HL-60 cells at a 2:1 effector to target ratio in an IncuCyte S3 Imager with the indicated treatments (30 nM each) for 48 hours. Dead HL-60 cells were measured using a caspase 3 / 7 reagent.

[0157] Figure 15A Quantification of the percentage of viable HL-60 tumor target (CellTrace Far Red / Caspases 3 / 7) normalized to the target alone and the 0 hour time point is shown. Readings were taken every 30 minutes over 48 hours. Representative of three separate experiments. The first from the top corresponds to no treatment, the second from the top corresponds to treatment with CLEC12AscFv, the third from the top corresponds to treatment with IL-15, and the fourth from the top corresponds to treatment with CLEC12A TriKE. The lowest percentage of viable HL-60 cells was observed in the presence of CLEC12A TriKE.

[0158] Figure 15BRepresentative images after 0, 18, and 36 hours show HL-60 target cells (larger cells) and NK cells (smaller cells). After 0 hours, there were almost no dead cells for all indicated treatment conditions. After 18 and 36 hours, there were almost no dead HL-60 cell clusters across all no-treatment and CLEC12A scFv conditions, with some dying HL-60 cell clusters present for IL-15 treatment and more dying cell clusters in the case of CLEC12A TriKE treatment.

[0159] These data indicate that the CD16-IL15-CLEC12A TriKE induced killing of AML targets, including THP-1 and HL-60 target cells.

[0160] Example 8

[0161] This example demonstrates in vitro CD16-IL15-CLEC12A TriKE-induced killing of primary AML blast targets.

[0162] Enriched NK cells were incubated with primary AML blasts at a 2:1 effector-to-target ratio in the presence of equimolar concentrations of IL-15, CLEC12A scFv, CLEC12A TriKE, or CD33 TriKE. Figure 8A Shown is the gating scheme for identifying AML blasts using the FlowJo Analyzer. Figure 8B Shown is the percentage killing of AML blasts after gating on blasts after 48 hours as assessed by live / dead markers. + CD3 - Surface CD107a expression was assessed on NK cells to estimate degranulation ( Figure 8C ) and intracellular IFNg to estimate inflammatory cytokine production ( Figure 8D ). Statistics reflect significant differences between groups as calculated using one-way ANOVA, *P<0.05, **P<0.005, N=10. The percentage of dead AML blasts and the percentage of CD107a and IFNg staining were significantly higher when incubated with CLEC12A TriKE compared to incubation with IL-15 or incubation with CLEC12AscFv ( Figure 8B -D).

[0163] In other experiments, primary AML blasts (SSCh low, CD45 intermediate, CD117+, CD14-, CD34+) were evaluated for expression of CD33 and CLEC12A using flow cytometry ( Figure 12A). Cells expressed CD33, CLEC12A, or CD33 and CLEC12A, as indicated.

[0164] Enriched NK cells (CD56+, CD3-) from healthy donors (n=10) were incubated with primary AML blasts with the indicated treatments (30 nM) to estimate CD107a expression as a measure of degranulation ( Figure 12B ) and intracellular IFNg production ( Figure 12C ) markers. CLEC12A TriKE and CD33 TriKE induced degranulation and IFNg production as shown. Target cell killing ( Figure 12D ). Treatment with IL-15, CLEC12A TriKE, and CD33 TriKE resulted in killing of AML blasts, as shown, with CLEC12A TriKE-mediated killing being greater.

[0165] The proportion of AML blasts in different groups (based on CD33 and CLEC12A expression) was tracked over 48 hours to assess the specificity of 1615CLEC12A TriKE compared to 1615CD33 TriKE. Figure 12E ). The bars showing % survival of AML blasts correspond to the following conditions (from the top of each bar): (i) for no treatment, CLEC12A+CD33+ and CLEC 12a+CD33-; (ii) for IL-15, CLEC12A+CD33+, CLEC12A+CD33-, and CLEC 12a-CD33-; (iii) for CLEC12AscFv, CLEC12A+CD33+, CLEC12A+CD33-, CLEC12A-CD33+, and CLEC12A-CD33-; (iv) for CLEC12ATriKE, CLEC12A+CD33+, CLEC12A-CD33+, and CLEC12A-CD33-; (v) for CD33 TriKE, CLEC12A+CD33+ and CLEC12A+CD33-. These data confirm the specificity of 1615CLEC12A TriKE compared to 1615CD33 TriKE.

[0166] In other experiments, enriched NK cells (CD56+, CD3-) from healthy donors (n=5) were incubated with bone marrow samples from AML patients with the indicated treatments (30 nM) to assess killing of cancer stem cells (SSCh low, CD45 medium, CD34+, CD38-) in a 4-hour assay at an effector / target ratio of 2:1 ( Figure 12F ). Representative flow chart showing the killing of CLEC12A and CD33 positive cancer stem cells.

[0167] Figure 12G Composite data from the cancer stem cell killing assay are shown, showing the percentage of cancer stem cells present at the end of the assay. Differences for the 1615CLEC12A group were calculated using a one-way analysis of variance (ANOVA) with repeated measures. Error bars represent + / - standard error of the mean. Statistical significance was determined as * P, .05, ** P, .01, *** P, .001 and **** P, .0001. Treatment with CLEC12A TriKE resulted in a significant decrease in the percentage of LSCs compared with no treatment.

[0168] To examine primary AML blasts as target cells for CLEC12A TriKE-mediated killing, a gating strategy was employed. Figure 16A Primary AML blasts (n=5) were then incubated with the indicated treatments (30 nM) for 48 h to estimate target cell killing using flow cytometry and live / dead markers ( Figure 16B ). The percentage of dead AMP protocells after 48 hours is shown.

[0169] These data indicate that 1615CLEC12A TriKE induced a slight increase in killing of primary AML blasts in patient samples during blast crisis in which NK cells were restricted.

[0170] Example 9

[0171] This example describes 1615CLEC12A TriKE-mediated restriction of tumor growth in vivo.

[0172] Figure 13A Schematic diagram of HL-60luc mouse experiment is shown. The model was established by conditioning NSG mice (225 cGy) and then intravenously injecting HL-60luc cells (7.5×105 cells / mouse). Three days later, 1×105 cells activated overnight with 10 ng / ml of IL-15 were infused. 6 Normal human donor NK cells (calculated based on CD3 / CD19 product of magnetic depletion). In the following 3-week study, 1615CLEC12A TriKE or 161533TriKE (20 μg) MTWThF were administered (total 15 doses), and the control group received only HL-601uc cells.

[0173] Quantification of luminescence from the four treatment groups on days 7, 14, and 21 after NK infusion was performed. Figure 13B Each dot represents a different mouse, and the bars represent the mean + / - standard deviation. Differences for the 1615CLEC12A group were calculated using one-way analysis of variance (ANOVA) without matched comparisons. Statistical significance was determined as *P, .05, ***P, .001, and ****P, .0001. Figure 13C Shown are individual mouse photoluminescence (dark areas) after a 2-minute exposure on days 7, 14, and 21. CLEC12A TriKE treatment resulted in reduced tumor burden at all time points examined compared to the HL60 control. In addition, a significant reduction in tumor burden was observed after CLEC12A TriKE treatment on day 21 relative to the NK cell control treatment.

[0174] Figure 13D Schematic diagram of the pdx mouse experiment is shown. NSG SGM3 mice were conditioned (125 cGy) and then intravenously injected with HL-60luc cells (7.5×10 5 The model was established by allowing the tumor to grow until at least 1% of AML blasts were present in the blood. 1×10 cells activated overnight with 10 ng / ml of IL-15 were then infused. 6 Normal human donor NK cells (calculated based on CD3 / CD19 product of magnetic depletion). In the following 3-week study, 1615CLEC12ATriKE or 161533TriKE (20 μg) MTWThF was administered (15 doses in total), and the control group received NK cells but did not receive treatment. Mice were sacrificed on day 21, and the percentage of AML blasts (CD45 medium, CD33+) in the bone marrow from the femur was calculated by flow cytometry ( Figure 13E Each dot represents a different mouse. Bone marrow samples were counted by flow cytometry ( Figure 13F ) and peripheral blood ( Figure 13G ) in the 1615CLEC12A group. The events were collected over 60 seconds and the number of human NK cell events was calculated. A representative dot plot showing the number of NK (CD56+CD3-) cell events within the CD45+ gate is shown. The differences for the 1615CLEC12A group were calculated using a one-way analysis of variance (ANOVA) without matched comparisons. The error bars represent the mean + / - standard deviation. Statistical significance was determined as *P, .05, ***P, .001, and ****P, .0001. The results showed that NK+CLEC12A TriKE treatment significantly reduced the percentage of primary AML blasts compared to tumors alone ( Figure 13EFurthermore, NK+CLEC12A TriKE treatment resulted in bone marrow ( Figure 13F ) and peripheral blood ( Figure 13G ) showed a significant increase in the percentage of NK cells.

[0175] Together, these results demonstrate that 1615CLEC12A TriKE limits tumor growth in vivo.

[0176] Example 10

[0177] This example describes the analysis of stem and progenitor cells.

[0178] use Figure 17 The gating strategy shown identifies cancer stem cells in bone marrow samples. Figure 19 The following table shows the determination of different CD34 pos Gating strategy for progenitor cell subsets. Figure 19 The gating strategy shown was used for the analysis Figure 18A The cell populations shown.

[0179] Figure 18A CD34 in bone marrow from two representative healthy donors is shown. pos Expression of CLEC12A and CD33 in the progenitor compartment. HSC: hematopoietic stem cell, MPP: multipotent progenitor, LMPP: lymphoid-primed multipotent progenitor, CLP: common lymphoid progenitor, CMP: common myeloid progenitor, GMP: granulocyte-macrophage progenitor, MEP: megakaryocyte-erythroid progenitor. CLEC12A expression was observed in the CMP, GMP, and MEP populations, but was relatively low in the HSC, MPP, CLP, and LMPP populations. In addition, with the exception of the GMP population, CLEC12A was found to be at lower levels than CD33. After treatment with 1615CLEC12A TriKE or 161533TriKE, burst forming unit erythroid (BFU-E) colonies and colony forming unit erythroid (CFU-E) colonies were counted ( Figure 18B ). Larger numbers of BFU-E and CFU-GM colonies were observed after treatment with 1615CLEC12A TriKE compared to treatment with 161533 TriKE.

[0180] The data showed that CLEC12A is differentially expressed in normal donor stem and progenitor cell populations, and that treatment with 161533 TriKE reduced stem cell formation and / or differentiation compared to treatment with CLEC12A TriKE. Without being limited by theory, this suggests that while CLEC12A can be used to target leukemic stem cells, normal hematopoietic reconstitution should be allowed, whereas CD33 targeting is more likely to affect reconstitution. In other words, CLEC12A targeting should have fewer off-target effects with respect to normal bone marrow reconstitution.

[0181] In summary, the above data show that the CD16-IL15-CLEC12A TriKE specifically binds to target cells expressing CLEC12A, promotes the proliferation of NK cells, enhances the function of NK cells, promotes the killing of AML cell lines in the Incucyte zoom assay, and induces the killing of primary AML and MDS blasts.

[0182] Example 11

[0183] This example illustrates the generation of TetraKEs targeting CLEC12A and a second target or tumor antigen.

[0184] TetraKE (tetramer) molecules containing more than one targeting domain can be designed. As an example, a TetraKE can contain an NK engagement domain, an NK activation domain, and two targeting domains. Any NK engagement domain and NK activation domain described herein can be used. For example, the targeting domains can target different targets or tumor antigens. Any combination of targets or tumor antigens can be included in a TetraKE. For example, a first targeting domain can bind to CLEC12A, while a second targeting domain can bind to another target or tumor antigen.

[0185] Any target or tumor antigen described herein can be included in a TetraKE having a first targeting domain that binds to CLEC12A, comprising, for example, a second targeting domain that binds to CD133, CD20, HER2, CEA, EpCAM, VEGF-A, EGFR, CD33, integrin αVβ3, CD51, CD152, CD125, CTAA16.88, MUC1, CD19, CD22, CD38, mesothelin, ROR1, CSPG4, SS1 or IGFR1, NKG2C, BCMA, APRIL, B7H3 and PSMA, or a viral antigen derived from EBV, HBV, HCV and / or HPV. In addition, the TetraKE domains can be operably linked to each other using flanking or linker sequences as described herein. Exemplary TetraKEs comprise a compound having a portion that selectively binds to CD16, a NK activation domain comprising IL-15, a first targeting domain that selectively binds to CLEC12A, and a second targeting domain that selectively binds to CD33.

[0186] SEQ ID NO.: 1

[0187] MKWVTFISLLFLFSSAYSQVQLVESGGGLVQPGGSLRLSCAASGLTFSSYNMGWFRQAPGQGLEAVA

[0188] SITWSGRDTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAANPWPVAAPRSGTYWGQG

[0189] TLVTVSSSSGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVT

[0190] AMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIV

[0191] QMFINTSGSTSGSGKPGSGEGSTKGQVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPP

[0192] GKGLEWIGEIYHSGSPDYNPSLKSRVTISVDKSRNQFSLKLSSVTAADTAVYYCAKVSTGGFFDYWG

[0193] QGTLVTVSSGGGGSGGGGSGGGGSEIELTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP

[0194] KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGPGTKVEIK

[0195] SEQ ID NO.:2

[0196] MKWVTFISLLFLFSSAYSQVQLVESGGGLVQPGGSLRLSCAASGLTFSSYNMGWFRQAPGQGLEAVA

[0197] SITWSGRDTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAANPWPVAAPRSGTYWGQG

[0198] TLVTVSSSGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVT

[0199] AMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIV

[0200] QMFINTSGSTSGSGKPGSGEGSTKGQVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPP

[0201] GKGLEWIGEIYHSGSPDYNPSLKSRVTISVDKSRNQFSLKLSSVTAADTAVYYCAKVSTGGFFDYWG

[0202] QGTLVTVSSGGGGSGGGGSGGGGSEIELTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP

[0203] KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGPGTKVEIKVDEHHH

[0204] HHHHHHH

[0205] SEQ ID NO.:3

[0206] VDEHHHHHHHHHH

[0207] SEQ ID NO.:4

[0208] QVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSPDYNPSLKSR

[0209] VTISVDKSRNQFSLKLSSVTAADTAVYYCAKVSTGGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGS

[0210] EIELTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGT

[0211] DFTLTISSLQPEDFATYYCQQSYSTPPTFGPGTKVEIK

[0212] SEQ ID NO.:5

[0213] atgaagtgggtaacctttatttcccttctttttctctttagctcggcttattcccaggtgcagctggtggagtctgggggaggcttggtgcagcctgggggctctctg

[0214] agactctcctgtgcagcctctggcctcaccttcagtagctataacatgggctggttccgccaggctccagggcaaggccttgaggctgtagcatctattacctg

[0215] gagtggtcgggacacattctatgcagactccgtgaagggccgattcaccatctccagagacaactccaagaacactctctatctgcaaatgaacagcctgcgc

[0216] gcggaggacacggccgtttattattgtgctgcaaacccctggccagtggcggcgccacgtagtggcacctactggggccaagggaccctggtcaccgtctc

[0217] ctcatctggcggcggcggttctggtggaggaggtagtggggggggaggaagcggagggggtggctcagggaactgggtgaatgtaataagtgatttgaa

[0218] aaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttggag

[0219] ttacaagttatttcacttgagtccggagatgcaagtattcatgatacagtagaaaatctgatcatcctagcaaacaacagtttgtcttctaatgggaatgtaacaga

[0220] atctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttctggcagtacca

[0221] gcgggtcagggaaacctggcagtggggaaggttccacaaaaggtcaagtacaactccaggagtccgggccagggttggtcaagccatccgagacgctta

[0222] gtttgacctgtgttgtcagcggaggctctatatcatcttcaaactggtggtcttgggtacggcaaccaccgggcaaggggctcgaatggatcggggaaatctac

[0223] cactccggaagccccgactataatccgtcactgaagagcagagtcactatatccgtggacaagagcagaaaccaattttctcttaagctctcctcagtgacagc

[0224] agcagatacagcggtctattattgtgccaaggtatcaacaggcggattcttcgattattggggacagggcactttggttacggtttcttctggaggcgggggaa

[0225] gtggtggaggggggtctgggggaggtggctcagaaatcgaacttacgcagtcaccctcctccctctcagcatccgtaggtgacagagttacgataacctgta

[0226] gagcaagtcaatccatttctagctaccttaactggtatcagcaaaaacctgggaaagcccccaagctgcttatctatgcggcatcctccctccaaagtggagttc

[0227] ccagtcggttcagtggttccggctcagggactgactttaccctcacaatcagctcattgcaaccagaggactttgcaacgtattactgtcagcaaagctactcaa

[0228] cgccgcctacgttcggtcccggaaccaaagttgagattaaagtagacgaacaccatcatcatcatcaccatcaccaccattga

[0229] SEQ ID NO.:6

[0230] atgaagtgggtaacctttatttcccttctttttctctttagctcggcttattcccaggtgcagctggtggagtctgggggaggcttggtgcagcctgggggctctctg

[0231] agactctcctgtgcagcctctggcctcaccttcagtagctataacatgggctggttccgccaggctccagggcaaggccttgaggctgtagcatctattacctg

[0232] gagtggtcgggacacattctatgcagactccgtgaagggccgattcaccatctccagagacaactccaagaacactctctatctgcaaatgaacagcctgcgc

[0233] gcggaggacacggccgtttattattgtgctgcaaacccctggccagtggcggcgccacgtagtggcacctactggggccaagggaccctggtcaccgtctc

[0234] ctcatctggcggcggcggttctggtggaggaggtagtggggggggaggaagcggagggggtggctcagggaactgggtgaatgtaataagtgatttgaa

[0235] aaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttggag

[0236] ttacaagttatttcacttgagtccggagatgcaagtattcatgatacagtagaaaatctgatcatcctagcaaacaacagtttgtcttctaatgggaatgtaacaga

[0237] atctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttctggcagtacca

[0238] gcgggtcagggaaacctggcagtggggaaggttccacaaaaggtcaagtacaactccaggagtccgggccagggttggtcaagccatccgagacgctta

[0239] gtttgacctgtgttgtcagcggaggctctatatcatcttcaaactggtggtcttgggtacggcaaccaccgggcaaggggctcgaatggatcggggaaatctac

[0240] cactccggaagccccgactataatccgtcactgaagagcagagtcactatatccgtggacaagagcagaaaccaattttctcttaagctctcctcagtgacagc

[0241] agcagatacagcggtctattattgtgccaaggtatcaacaggcggattcttcgattattggggacagggcactttggttacggtttcttctggaggcgggggaa

[0242] gtggtggaggggggtctgggggaggtggctcagaaatcgaacttacgcagtcaccctcctccctctcagcatccgtaggtgacagagttacgataacctgta

[0243] gagcaagtcaatccatttctagctaccttaactggtatcagcaaaaacctgggaaagcccccaagctgcttatctatgcggcatcctccctccaaagtggagttc

[0244] ccagtcggttcagtggttccggctcagggactgactttaccctcacaatcagctcattgcaaccagaggactttgcaacgtattactgtcagcaaagctactcaa

[0245] cgccgcctacgttcggtcccggaaccaaagttgagattaaatga

[0246] SEQ ID NO.:7

[0247] caagtacaactccaggagtccgggccagggttggtcaagccatccgagacgcttagtttgacctgtgttgtcagcggaggctctatatcatcttcaaactggtg

[0248] gtcttgggtacggcaaccaccgggcaaggggctcgaatggatcggggaaatctaccactccggaagccccgactataatccgtcactgaagagcagagtc

[0249] actatatccgtggacaagagcagaaaccaattttctcttaagctctcctcagtgacagcagcagatacagcggtctattattgtgccaaggtatcaacaggcgg

[0250] attcttcgattattggggacagggcactttggttacggtttcttctggaggcgggggaagtggtggaggggggtctgggggaggtggctcagaaatcgaactt

[0251] acgcagtcaccctcctccctctcagcatccgtaggtgacagagttacgataacctgtagagcaagtcaatccatttctagctaccttaactggtatcagcaaaaa

[0252] cctgggaaagcccccaagctgcttatctatgcggcatcctccctccaaagtggagttcccagtcggttcagtggttccggctcagggactgactttaccctcac

[0253] aatcagctcattgcaaccagaggactttgcaacgtattactgtcagcaaagctactcaacgccgcctacgttcggtcccggaaccaaagttgagattaaa

[0254] SEQ ID NO.:8

[0255] QVQLVESGGGLVQPGGSLRLSCAASGLTFSSYNMGWFRQAPGQGLEAVASITWSGRDTFYADSVKG

[0256] RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAANPWPVAAPRSGTYWGQGTLVTVSS

[0257] SEQ ID NO.:9

[0258] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIIL

[0259] ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0260] SEQ ID NO.:10

[0261] MKWVTFISLLFLFSSAYS

[0262] SEQ ID NO.:11

[0263] SGGGGSGGGGSGGGGSGGGGSG

[0264] SEQ ID NO.:12

[0265] GSTSGSGKPGSGEGSTKG

[0266] SEQ ID NO.:13

[0267] PSGQAGAAASESLFVSNHAY

[0268] SEQ ID NO.:14

[0269] EASGGPE

[0270] SEQ ID NO.:15

[0271] GGGGSGGGGS

[0272] SEQ ID NO.:16

[0273] MGWSCIILFLVATATGVHSS

[0274] SEQ ID NO.:17

[0275] MGWSCIILFLVATATGVHS

[0276] SEQ ID NO.:18

[0277] EVQLVESGGELVQAGGSLRLSCAASGLTFSSYNMGWFRRAPGKEREFVASITWSGRDTFYADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCAANPWPVAAPRSGTYWGQGTQVTVSSVDE

[0278] SEQ ID NO. describe SEQ ID NO.: 1 CLEC12A TriKE SEQ ID NO.: 2 CLEC12A TriKE with His tag and spacer SEQ ID NO.: 3 His tag and spacer SEQ ID NO.: 4 CLEC12A targeting domain SEQ ID NO.: 5 DNA encoding CLEC12A TriKE with a His tag and a spacer SEQ ID NO.: 6 DNA encoding CLEC12ATriKE SEQ ID NO.: 7 DNA encoding the CLEC12A targeting domain SEQ ID NO.:8 Humanized Cam16 SEQ ID NO.: 9 Wild-type IL-15 SEQ ID NO.: 10 signal peptide SEQ ID NO.: 11 connector SEQ ID NO.: 12 connector SEQ ID NO.: 13 connector SEQ ID NO.: 14 connector SEQ ID NO.: 15 connector SEQ ID NO.: 16 signal peptide SEQ ID NO.: 17 signal peptide SEQ ID NO.: 18 Non-humanized Cam16

[0279] Any and all references and citations to other documents, such as patents, patent applications, patent publications, journals, books, treatises, web content, throughout this disclosure are hereby incorporated by reference in their entirety for all purposes.

[0280] Although the present invention has been described in detail with reference to certain embodiments of the present invention, it should be understood that modifications and variations are encompassed within the spirit and scope of the present invention. Therefore, the present invention is limited only by the appended claims.

Claims

1. A compound comprising: NK engagement domain; a NK activation domain operably linked to the NK engagement domain; and A targeting domain that selectively binds to a target cell and is operably linked to the NK activation domain and the NK engagement domain, wherein the targeting domain selectively binds to CLEC12A.

2. The compound of claim 1, wherein the NK engaging domain comprises a portion that selectively binds to CD16.

3. The compound of claim 1, wherein the NK engaging domain portion comprises an antibody or binding fragment thereof or a nanobody.

4. The compound of claim 3, wherein the antibody binding fragment comprises scFv, F(ab)2 or Fab.

5. The compound of claim 3, wherein the antibody or binding fragment thereof or the Nanobody is human or humanized.

6. The compound of claim 3, wherein the antibody or binding fragment thereof or the nanobody is of Camelidae.

7. The compound of claim 1, wherein the NK activation domain comprises IL-15 or a functional fragment thereof.

8. The compound according to claim 7, wherein the IL-15 comprises the amino acid sequence of SEQ ID NO: 9 or a functional variant thereof.

9. The compound of claim 1, wherein the targeting domain portion comprises an antibody or binding fragment thereof or a nanobody.

10. The compound of claim 9, wherein the antibody binding fragment comprises scFv, F(ab)2, or Fab.

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